建兰花叶病毒CP基因的原核表达及抗血清制备

通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒。设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a（＋）中构建相应的原核表达载体。目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清。Westernblot检测结果表明,抗血清与诱导表达的CyMV外壳蛋白发生特异性反应。间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1∶51200,最佳工作浓度为1∶1000,病汁液灵敏度为0.39mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应。

Prokaryotic Expression and Antiserum Preparation of the Coat Protein of Cymbidium Mosaic Virus

Cymbidium mosaic virus (CyMV) is one of the most important and worldwide viruses attacking orchids. This virus causes the symptoms of mosaic,chlorosis,necrosis and malformation in the orchids,and has a high economic impact to the orchid industry. Cattleya plants contracted with a disease were collected as samples from Zhangzhou,Fujian,and were identified to be infected with Cymbidium mosaic virus by using ID -ELISA and electronic microscopy assay. One pair of specific primers was designed for amplification of the coat protein(CP) gene from the samples infected with CyMV. The open reading frame encoding CP of CyMV isolate obtained from Zhangzhou,Fujian is 672 bp,encoding a 23.6 ku protein with 223 aa. The expected CP gene was then inserted into the pET-29a(+)vector for prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According the result of ID-ELISA analysis,specific rabbit anti-CyMV serum was prepared with a high titre of 1:51 200,a working concentration of 1:1 000 and sap sensitivity of 0.39 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of CyMV. There were no cross reactions between the antiserums and 11 species of homologous or heterologous viruses such as tobacco mosaic virus.